Arfs (ADP ribosylation factors) are members of a large family of GDP/GTP switch proteins that are a key element in the control of vesicle traffic between membranes of the endoplasmic reticulum, the Golgi, and the plasma membrane. Vesicle trafficking is a process essential to the normal posttranslational modification of newly synthesized proteins and the proper export of those proteins. Certain diseases have been linked to variations in vesicle traffic, and certain pathogens are known to highjack the vesicle trafficking process in order to create environments in which they can replicate. These observations make understanding control of vesicle traffic important to the development of new approaches for combating disease. Arfs affect control of vesicle traffic through reversible membrane association in response to exchange of GDP for GTP, but this is not done in isolation;Arfs must interact with various coat proteins to recruit cargo, nucleotide exchange is facilitated by interaction with various Guanine nucleotide exchange factors (GEFs), and association with membranes is affected by membrane-localized lipids such as the phosphatidylinositide, PI(4,5)P2. The particular focus of the proposed research will be on structural responses of human Arf1 on interaction with membrane mimetics, a GEF from Legionella pneumophila called RalF, and a construct of the Alzheimers associated coat protein, MINT2. The work will also include a structural investigation of the interaction of MINT2 with beta amyloid precursor protein (APP). In the process, new NMR methods based on residual dipolar couplings and other anisotropic spin interactions such as chemical shift anisotropy offsets are developed. These methods allow collection of structural information on the assembly of complex structures, including those that are organized at a membrane surface. Their development will have an impact on investigation of other membrane associated systems. Importance to public health: The proposed studies will result in structural models of several protein complexes intimately involved in human disease. These include the Arf1-RalF interaction important to Legionnaire's disease and the Arf1-MINT2-APP interaction important to Alzheimer's disease. Structural information can aid in the development of new drugs to combat these diseases.